This was the third year for this project, using biochemical methods to study conformational changes in ClC-type chloride channel proteins. The ClC family of chloride-conducting ion channels is involved in a host of biological processes; these channels maintain the resting membrane potential in skeletal muscle, modulate excitability in central neurons, and are involved in the homeostasis of pH in a variety of intracellular compartments. Despite their physiological importance, the mechanisms by which these channels function are poorly understood. We are attempting to understand the functional properties of these proteins by examining the function of several family members, including both eukaryotic and prokaryotic homologs. In this project, we are using the prokaryotic ClC's for biochemical studies of conformational changes upon activation of the transport process. Toward this goal, we have constructed a series of cysteine mutants in a critical region of the transport protein and can covalently attach fluorescent probes to these sites. We then examine the fluorescence properties these proteins after reconstitution into liposomes; we find that there are dramatic and reversible changes in fluorescence upon activation of transport at low pH, presumeably reflecting a significant conformational change. Various controls demonstrate no change in fluorescence of the isolated fluorophore with pH, and that we can specifically modify the cysteine of interest. We have established that the conformational change is sensitive to chloride and protons, the two native subtrates of the transporter. Fluorescence changes persist in transporters lacking a critical 'gating glutamtate,' suggesting that other titratable groups contribute to the transport process. In addition, the labeled mutant transporters are active, showing that the fluorescent probe does not disrupt the transport process. We are currently examining further mutants to establish the extent of the conformational change, as well as double mutants designed to test hypotheses regarding its mechanism.